Sugar Extract

ABSTRACT

This invention relates to novel extracts from sugar cane and sugar beet molasses and the characterisation of those extracts. The extracts are enriched in hydrophobic compounds including polyphenols, in levels 5 to 10 fold higher than found in molasses itself Methods for extracting the extract are also described, together with new uses for the extracts as food ingredients, food modifiers and therapeutic substances.

FIELD OF THE INVENTION

The invention relates to extracts produced from sugar cane and sugarbeet waste processing stream products having desirable properties andhealth benefits. More particularly the invention relates to hydrophobicextracts obtained from molasses, methods of producing the extracts, usesof the extracts, and products containing the extracts.

BACKGROUND OF THE INVENTION

Sugar is a common carbohydrate sourced from sugar cane and sugar beetused in food because of its sweet taste. Ordinary table sugar (sucrose)is a disaccharide made up of one molecule of glucose bound by aα-1,2-glycoside to one molecule of fructose. Table sugar is 99.5%sucrose, the most biologically abundant disaccharide. Saccharides aresimple carbohydrates classified as monosaccharides, oligosaccharides orpolysaccharides depending upon their structure. Sucrose is sourced fromboth sugarcane and beets.

i) Sugar Processing

The processing steps required to produce white sugar result ingeneration of a number of byproducts, most of which are consideringwaste products with little or no nutritional value or use in humanapplications.

After being mechanically harvested, sugar cane is transported to a milland crushed between serrated rollers. The crushed sugar cane is thenpressed to extract the raw sugar juice, while the bagasse (leftoverfibrous material) is used for fuel. The raw juice is then heated to itsboiling point to extract any impurities and lime and bleaching agentsare added and mill mud is removed. The raw juice is further heated undervacuum to produce bulk sugar crystals and a thick syrup known asmolasses. The two are separated by a centrifuge and the molasses wastestream is collected for use as a low-grade animal feedstock. The bulksugar crystals are further refined to increase their purity.

The bulk sugar crystals from the above process are further refined toproduce the many commercially available sugar products. The bulk sugarcrystals are mixed with a hot concentrated syrup to soften the outercoating on the crystals. The crystals are recovered by centrifuge andthen dissolved in hot water. This sugar liquor is then further purifiedby carbonation or phosfloatation, filtration, decolourisation and thenseeded with fine sugar crystals. Once the crystals have grown to therequisite size, the crystals are separated from the syrup by centrifuge,dried, graded and then packaged. There may be several repetitions ofrecovering sugar crystals from the sugar liquor. The dark sugar syrupwhich is left after all of the sugar crystals have been recovered isalso called molasses.

Approximately 70% of the world's sugar comes from sugar cane and about30% comes from sugar beets Similar processes are used to manufacturesugar products from sugar beets. However, it is a single step ratherthan two step process.

The processing starts by slicing the beets into thinstrips/chips/cossettes. This process increases the surface area of thebeet to make it easier to extract the sugar. The extraction takes placein a diffuser where the beet is kept in contact with hot water and theresultant sugar solution is referred to as the juice. The exhausted beetslices from the diffuser are then pressed to squeeze as much juice aspossible out of them. The pressed beet, by now a pulp, is sent to dryingplant where it is turned into pellets which form an importantconstituent of some animal feeds. The juice is then cleaned up before itcan be used for sugar production and the non-sugar chemicals are removedin a process called carbonation (milk of lime (calcium hydroxide) andcarbon dioxide gas). The calcium carbonate (chalk) which forms traps thenon-sugar chemicals and is removed (called mud) in the clarifier. Oncethis is done the sugar liquor is concentrated until sugar crystals form.Once the crystals have grown the resulting mixture of crystals andmother liquor is spun in centrifuges to separate the two. The crystalsare then given a final dry with hot air before being packed and/orstored ready for dispatch. The final sugar is white and ready for use.Because one cannot get all the sugar out of the juice, there is a sweetby-product made: beet molasses. This is usually turned into a cattlefood or is sent to a fermentation plant such as a distillery wherealcohol is made.

ii) Polyphenols, Polyphenol Glycosides and Phenolic Acids

Polyphenols (compounds with two or more phenol groups) are a class ofphytochemicals found in a variety of sources including wine, grapes,cocoa and sugar cane and sugar beet. Natural polyphenols can range fromsimple molecules such as phenolic acids to large highly polymerizedcompounds such as tannins Polyphenols (or phenolics) all have a commonbasic chemical component, that is, a phenolic ring structure. There areat least 8000 identified polyphenols in a number of subcategories, suchas anthocyanins and catechins. Polyphenols can exist in their free form,or as polyphenol glycosides.

Conjugated forms of polyphenols are the most common, where various sugarmolecules, organic acids and lipids (fats) are linked with the phenolicring structure. Despite having a common phenolic ring structure,differences in the conjugated chemical structure, size and othersubstituents account for different chemical classifications andsignificantly, variation in the modes of action and health properties ofthe various compounds.

Phenolic acids are simple molecules such as caffeic acid, vanillin, andcoumaric acid. Phenolic acids form a diverse group that includes thewidely distributed hydroxybenzoic and hydroxycinnamic acids (despite thelatter two only having one phenolic ring). Hydroxycinnamic acidcompounds (p-coumaric, caffeic acid, ferulic acid) occur most frequentlyas simple esters with hydroxy carboxylic acids or glucose, while thehydroxybenzoic acid compounds (p-hydroxybenzoic, gallic acid, ellagicacid) are present mainly in the form of glucosides. Coffee isparticularly rich in bound phenolic acids, such as caffeic acid, ferulicacid, and p-coumaric acid.

Reference to any prior art in the specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any otherjurisdiction or that this prior art could reasonably be expected to beascertained, understood and regarded as relevant by a person skilled inthe art.

SUMMARY OF THE INVENTION

The invention relates to hydrophobic extracts obtained from molasses,methods of producing the extracts, uses of the extracts, and productscontaining the extracts. In one aspect of the invention, there isprovided a molasses extract with a high relative abundance ofhydrophobic compounds including polyphenols. Ie a molasses extractenriched with hydrophobic compounds including polyphenols compared tomolasses itself. More particularly, there is provided a molasses extractwith a high relative abundance of hydrophobic compounds includingpolyphenols wherein the extract comprises

-   -   at least 9000 mg CE/100 g of hydrophobic polyphenols in a        mixture of free form polyphenols selected from apigenin,        catechin, catechin gallate, epicatechin, kaempherol, luteolin,        quercetin, tricin, myricetin and diosmetin; polyphenol        glycosides selected from diosmin, tricin-7-O-neohesperidoside,        orientin, vitexin, luteolin-8-C-(rhamnosylglucoside),        schaftoside, isoschaftoside, and rutin; and phenolic acids,        selected from caffeic acid, chlorogenic acid, p-coumaric acid,        ferulic acid, gallic acid, syringic acid, and vanillic acid;    -   trace elements selected from one or more of calcium, iron,        magnesium, manganese, potassium and sodium;    -   protein and other nitrogen-containing compounds; and    -   carbohydrates other than monosaccharides and sucrose        wherein the extract has less than 2 g of monosaccharides and        sucrose per 100 g of extract.

Preferably the hydrophobic polyphenols are present in an amount of atleast 18000 mg CE/100 g of extract, more preferably at least 21000CE/100 g extract, and the extract comprises a combination of all ofapigenin, catechin, catechin gallate, epicatechin, kaempherol, luteolin,quercetin, tricin, myricetin, diosmetin, diosmin,tricin-7-O-neohesperidoside, orientin, vitexin, luteolin- 8-C-(rhamnosylgluco side), schaftoside, isoschaftoside, rutin, caffeicacid, chlorogenic acid, p-coumaric acid, ferulic acid, gallic acid,syringic acid, and vanillic acid.

Extracts of the invention can be produced by contacting molasses with ahydrophobic polymeric adsorbent to bind compounds including polyphenolsin the molasses. In this aspect of the invention, there is provided amethod for producing a molasses extract with a high relative abundanceof hydrophobic compounds including polyphenols, comprising the steps of:

-   -   a. contacting a sample of molasses with a hydrophobic polymeric        adsorbent under conditions sufficient to enable binding of        compounds to the adsorbent; and    -   b. eluting the bound compounds

wherein the eluted product from step (b) has a high relative abundanceof hydrophobic compounds including polyphenols compared to the sample ofmolasses ie prior to step (a).

More particularly, there is provided a method for producing a molassesextract of the invention with a high relative abundance of hydrophobiccompounds including polyphenols comprising the steps of:

-   -   a. diluting the molasses to produce a 10 to 40% w/v aqueous        solution;    -   b. optionally filtering the diluted molasses produced in step        (a);    -   c. contacting the diluted molasses with a hydrophobic polymeric        adsorbent under conditions sufficient to enable binding of        compounds to the adsorbent and flow through of all other        compounds in the diluted molasses;    -   d. optionally passing the flow through from step (c) over the        hydrophobic polymeric adsorbent at least once;    -   e. optionally rising the hydrophobic polymeric adsorbent; and    -   f. eluting the compounds bound to the hydrophobic polymeric        adsorbent to produce the extract    -   wherein the compounds are eluted with 30 to 70% ethanol,        preferably 40% ethanol.

The extract has a high relative abundance of hydrophobic compoundsincluding polyphenols compared to the sample of molasses used as thestarting material.

There is also provided a molasses extract with a high relative abundanceof hydrophobic compounds including polyphenols obtained from the methodof the invention.

In another aspect of the invention, the extracts of the invention can beformulated in to a therapeutic composition for use in a number oftherapeutic methods. In one embodiment there is provided a method fordecreasing body fat and/or minimising fat accumulation in an animal byadministering a composition including a molasses extract having arelatively high abundance of hydrophobic compounds including polyphenolsin an amount effective to decrease total body fat and/or minimise fataccumulation of the animal.

In a further embodiment, there is provided a method of reducing energyabsorption and/or altering fat metabolism by administering a compositionincluding a molasses extract having a relatively high abundance ofhydrophobic compounds including polyphenols in an amount effective toreduce energy absorption and/or alter fat metabolism.

Methods of alleviating or reducing the severity of fatigue, and methodsof improving and elevating energy levels in an animal by administering acomposition including a molasses extract having a relatively highabundance of hydrophobic compounds including polyphenols in an amounteffective are also contemplated.

In a further embodiment, there is provided a method of improvingpostprandial satiety in an individual by administering a compositionincluding a molasses extract having a relatively high abundance ofhydrophobic compounds including polyphenols in an amount effective todecrease a desire to have further food.

As an alternative to administering a composition, the individual may beadministered the extract of the invention as part of a satiety inducingfood.

There is also provided use of an effective amount of a molasses extracthaving a relatively high abundance of hydrophobic compounds includingpolyphenols in the preparation of medicament for decreasing body fat,minimising fat accumulation, reducing energy absorption and/or alteringfat metabolism, improving and elevating energy levels in an animal, andimproving postprandial satiety.

In a further embodiment, there is provided an effective amount of amolasses extract having a relatively high abundance of hydrophobiccompounds including polyphenols for decreasing body fat, minimising fataccumulation, reducing energy absorption and/or altering fat metabolism,improving and elevating energy levels in an animal, and improvingpostprandial satiety.

The invention also provides a composition for decreasing body fat,minimising fat accumulation, reducing energy absorption and/or alteringfat metabolism, improving and elevating energy levels in an animal, andimproving postprandial satiety, the composition comprising as an activeingredient a molasses extract having a relatively high abundance ofhydrophobic compounds including polyphenols.

Another aspect of the present invention includes food productscomprising an extract according to the invention alone as the activeingredient or in combination with other active ingredients.

In yet another embodiment, there is provided a satiety inducing foodincluding a molasses extract having a relatively high abundance ofhydrophobic compounds including polyphenols.

In yet another embodiment, there is provided a pet food including amolasses extract having a relatively high abundance of hydrophobiccompounds including polyphenols, wherein the pet food is preferably forcompanion animals including cats, dogs and horses.

In each of these embodiments, the molasses extract of the invention ispreferably produced by the methods of the invention. These and otheraspects of the invention will now be described in greater detail.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: Mean differences (±SEM) in fat mass between the threeexperimental groups. * p<0.05—denotes significant difference from thecontrol group. (CON- control; 2%-2% extract; 4%-4% extract).

FIG. 1B: Mean differences (±SEM) in fat-free mass between the threeexperimental groups. (CON—control; 2%-2% extract; 4%-4% extract).

FIG. 2A: Mean (±SEM) final body weight for the three experimentalgroups. * p<0.05—denotes significant difference from the control group.(CON—control; 2%-2% extract; 4%-4% extract).

FIG. 2B: Mean (±SEM) DEXA body weight for the three experimental groups.** p<0.01—denotes significant difference from the control group. (CON-control; 2%-2% extract; 4%-4% extract).

FIG. 3: Glucose tolerance curve showing changes in blood glucoseconcentration (mmol/L) prior to and following administration of glucosesolution. (CON—control; 2%-2% extract; 4%-4% extract; AUC—area under thecurve).

FIG. 4A: Mean (±SEM) 24-hour energy expenditure of mice on theexperimental diets (CON- control; 2%-2% extract; 4%-4% extract).

FIG. 4B: Mean (±SEM) general locomotor activity of mice on theexperimental diets over a 24-hour period (CON- control; 2%-2% extract;4%-4% extract).

FIG. 5A: Mean (±SEM) Daily-excreted energy of mice on the experimentaldiets *** p<0.001—denotes significant difference from the control group.(CON- control; 2%-2% extract; 4%-4% extract).

FIG. 5B: Mean (±SEM) Digestibility, the percentage of energy that wasdigested from the diet consumed, for mice in each of the experimentalgroups. * p<0.05 *** p<0.001—denotes significant difference from thecontrol group. (CON- control; 2%-2% extract; 4%-4% extract).

FIGS. 5C, 5D, and 5E: Percentage differences (±SEM) of faecal matteranalyses for (C) lipid, (D) carbon and (E) nitrogen levels. *P<0.05,***P<0.001 denotes significance difference from control group.(CON—control; 2%-2% extract; 4%-4% extract).

FIG. 5F: Ratio of carbon to nitrogen in the faecal matter. *P<0.05,***P<0.001 denotes significance difference from control group. (CON-control; 2%-2% extract; 4%-4% extract).

FIG. 6A: Mean (±SEM) plasma leptin levels for mice in each of theexperimental groups. ** p<0.01—denotes significant difference from thecontrol group. (CON—control; 2%-2% extract; 4%-4% extract).

FIG. 6B: Mean (±SEM) plasma adiponectin levels for mice in each of theexperimental groups. (CON—control; 2%-2% extract; 4%-4% extract).

FIG. 7A: Mean (±SEM) fold change adipose adiponectin mRNA expression formice in each of the experimental groups. * p<.05 ** p<0.01—denotessignificant difference from the control group. (CON—control; 2%-2%extract; 4%-4% extract).

FIG. 7B: Mean (±SEM) fold change adipose PPARy mRNA expression for micein each of the experimental groups. ** p<0.01—denotes significantdifference from the control group. (CON—control; 2%-2% extract; 4%-4%extract).

FIG. 7C: Mean (±SEM) fold change adipose UCP2 mRNA expression for micein each of the experimental groups. (CON- control; 2%-2% extract; 4%-4%extract).

FIG. 7D: Mean (±SEM) fold change adipose FAS mRNA expression for mice ineach of the experimental groups. ** p<0.01—denotes significantdifference from the control group. (CON- control; 2%-2% extract; 4%-4%extract).

FIG. 8A. Mean (±SEM) fold change liver PPARc mRNA expression for mice ineach of the experimental groups. * p<0.05 *** p<0.001—denotessignificant difference from the control group. (CON- control; 2%-2%extract; 4%-4% extract).

FIG. 8B. Mean (±SEM) fold change liver UCP2 mRNA expression for mice ineach of the experimental groups. * p<0.05 *** p<0.001—denotessignificant difference from the control group. (CON- control; 2%-2%extract; 4%-4% extract).

FIG. 9: MRI analysis of the fat distribution in mice from the control,2% extract and 4% extract group.

FIG. 10: Shows the processing steps required to produce white sugar.

FIG. 11: Shows a method involving the step of passing molasses over ahydrophobic polymeric adsorbent to produce the extract of the inventionhaving a higher relative abundance of hydrophobic compounds includingpolyphenols compared to molasses that has not been exposed to apolymeric adsorbent.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Polyphenols are a class of phytochemicals found in a variety of sourcesincluding wine, grapes, cocoa, tea and sugar cane or sugar beet. Howeverthe inventors are the first to recognise that hydrophobic moleculesincluding particular hydrophobic polyphenols and their glycosides, aswell as phenolic acids, derived from sugar cane or beet waste streamproducts such as molasses, have specific health benefits.

i) High Polyphenol and Phenolic Acids Containing Molasses Extract andMethod of its Production

The inventors have found that the retentate obtained by subjectingmolasses derived from sugar cane or sugar beet to hydrophobic polymericadsorbance is enriched in hydrophobic molecules, particularlyhydrophobic polyphenols and their glycosides, as well as phenolic acids.Therefore, in one aspect of the invention, there is provided a molassesextract with a high relative abundance of hydrophobic compoundsincluding polyphenols.

By the phrase “hydrophobic compounds” it is intended to refer tocompounds with sufficient hydrophobicity to bind to a hydrophobicpolymeric adsorbant. Such compounds will have varying degrees of watersolubility, and as would be appreciated and understood by those skilledin the art, sufficient hydrophobicity would equate to a compound thatbinds to a hydrophobic adsorbent and must be eluted off. Ie the compoundwould not be removed by washing the adsorbent.

By the phrase “high relative abundance of hydrophobic compoundsincluding polyphenols” it is meant that the level of hydrophobiccompounds including polyphenols is enhanced or enriched, such that themolasses extract of the invention has a higher relative abundance ofhydrophobic compounds, and in particular polyphenols, compared tomolasses prior to processing of the molasses over a polymeric adsorbant.“Polyphenols” is intended to encompass free forms of polyphenols,polyphenol glycosides, and phenolic acids as referred to in more detailthroughout the specification.

Preferably, in relation to the hydrophobic polyphenols within theextract, the relative abundance of hydrophobic polyphenols is increasedby at least 5 fold, preferably by at least 7 fold, and most preferablyby at least 10 fold. This includes, as noted above, the free formpolyphenols, polyphenol glycosides and phenolic acids. For example,molasses, prior to being processed over a polymeric adsorbant hasapproximately polyphenols in an amount of 1800-2100 mg CE/100 g ofmolasses. The extract of the invention therefore preferably hashydrophobic polyphenols in an amount of at least 9000 mg CE/100 g, morepreferably at least 18000 mg CE/100 g, and most preferably at least21000 mg CE/100 g. “CE”, or “catechin equivalent” is a measure of totalpolyphenolic content, expressed as mg catechin equivalents/g crudematerial.

As used herein, the term “molasses” refers to the dark syrup which isleft behind after the bulk sugar crystals are collected in the sugarcane mill, the black syrup remaining after the sugar cane syrup has beencentrifuged for the last time in the refinery or beet molasses.Preferably, the molasses used is from the sugar cane mill.

The extracts of the present invention represent new products which areeconomically useful and can be used in a wide variety of applications.Accordingly, in another aspect of the invention, there is provided atherapeutic composition including a molasses extract with a highrelative abundance of hydrophobic compounds including polyphenols and apharmaceutically acceptable carrier, excipient or diluent.

The term “therapeutic composition” is a broad term which includesenteral and parenteral pharmaceutical preparations, nutraceuticals,supplements, functional foods and herbal preparations, some of which aredescribed in more detail below. Examples of suitable formulationsinclude tablets, powders, chewable tablets, capsules, oral suspensions,suspensions, emulsions or fluids, children's formulations, enteralfeeds, nutraceuticals, suppositories, nasal sprays, drinks and foodproducts. The carrier may contain any suitable excipients such as starchor polymeric binders, sweeteners, colouring agents, emulsifiers andcoatings. Preferably, the carrier is a food product or food ingredientsuch as sugar.

The therapeutic composition may be in any form appropriate foradministration to the subject. The therapeutic composition may beadministered topically, orally or by any other route of administration.

The compositions and methods of the present invention have applicationsin human medicine, the cosmetic and aesthetic industries, veterinarymedicine as well as in general, domestic and wild animal husbandry. Theterm “animal” as used herein therefore refers to any animal. Preferably,the animal is a mammal and more preferably a human An “animal” alsoincludes livestock species such as cattle, horses, sheep, pigs, goats,donkeys and poultry birds such as chickens, ducks, turkeys and geese ordomestic animals such as cats and dogs. An animal, regardless of whethera human or non-human animal, may also be referred to as an individual,subject, patient, host or recipient.

While there are methods in the art which subject sugar cane or sugarbeet products to extraction and purification processes, the skilledperson will appreciate that, depending on thepurification/extraction/treatment process used, the polyphenolcomposition of an end product will vary. For the first time, thisapplication describes a method involving the step of passing molassesover a hydrophobic polymeric adsorbe dance with the figure below) toproduce the extract of the invention having a higher relative abundanceof hydrophobic compounds including polyphenols compared to molasses thathas not been exposed to a polymeric adsorbant.

In one embodiment of this aspect of the invention there is provided amethod for producing a molasses extract with a high relative abundanceof hydrophobic compounds including polyphenols, comprising the steps of:

-   -   a. contacting a sample of molasses with a hydrophobic polymeric        adsorbent under conditions sufficient to enable binding of        compounds to the adsorbent; and    -   b. eluting the compounds

wherein the eluted product from step (b) has a high relative abundanceof hydrophobic compounds including polyphenols compared to the sample ofmolasses. In other words, the eluted product from step (b) has a highrelative abundance of hydrophobic compounds including polyphenolscompared to the sample of molasses prior to step (a). Preferably thehydrophobic compounds are eluted with 30 to 70% ethanol, most preferably40% ethanol.

As used herein, except where the context requires otherwise, the term“comprise” and variations of the term, such as “comprising”, “comprises”and “comprised”, are not intended to exclude further additives,components, integers or steps.

Preferably the hydrophobic polyphenols within the extract are at least 5to 10 fold higher than the molasses prior to processing over a polymericadsorbent, and are present in amounts of at least 9000 mg CE/100 g, morepreferably at least 18000 mg CE/100 g, and most preferably at least21000 mg CE/100 g of molasses extract.

Preferably the hydrophobic polymeric adsorbent is a polystyrene,non-ionic, hydrophobic, cross-linked polymer, containing both acontinuous polymer phase and continuous pore phase, and can be used inbatch or column form. This enables isolation of compounds of sufficienthydrophobicity to bind to the hydrophobic adsorbent, includingpolyphenols from the molasses. Preferably, large scale methods utilisecolumn (ie fixed bed) modes. A useful polymeric adsorbent for use in theinvention is Amberlite™ XAD16N™. It is preferred that the polymericadsorbent be food grade, particularly food grade for human use. A usefulfood grade polymeric adsorbent is AmberliteTM FPX66™.

Molasses from sugar cane or sugar beet is mixed with an aqueoussolution, preferably water, to form a diluted molasses solution.Preferably the molasses is a 10 to 40% w/v solution, and more preferablyapprox. a 20% w/v solution. Sediment and undissolved matter canoptionally be removed from the diluted molasses solution, either bycentrifugation, or more preferably, by filtration through an appropriatesized filter. 0.1 μM stainless steel filters are one such optionalthough the skilled person will be aware of other suitable filters inthe art. The diluted molasses is then contacted with the polymericadsorbent under conditions sufficient to enable binding of thehydrophobic compounds to the adsorbent. Typically the molasses sample isloaded on to the column at a flow rate of 2 to 6 L/min, preferably 4Lmin. It is also possible to define an extraction process by virtue ofthe length of time the sample is left in contact with the adsorbent.Preferably, in one embodiment, the diluted molasses is left in contactwith the polymeric adsorbant for a period of 30-120 minutes, andpreferably at least about 60 minutes, at ambient temperature. After thefirst flow through, the molasses can optionally be cycled across theresin in the column at least once more, preferably twice, for a total of3 cycles.

Following optional rinsing steps of the adsorbant with an aqueoussolution such as water, the retentate is then eluted, preferably with analcohol, to produce the molasses extract of the invention having a highrelative abundance of hydrophobic compounds including polyphenols. 30 to80% v/v ethanol is one suitable alcohol for use in eluting theretentate, passed over the column at a flow rate of 2 to 6 L/min,preferably 4 L/min. The elution step can also be defined by virtue oftime. Preferably, in one embodiment, the ethanol can be mixed with theadsorbent for a period of 2-15 minutes to ensure as much desorption ofthe hydrophobic compounds off the adsorbent as possible. 30 to 70%ethanol is used, preferably 40% ethanol.

The extract obtained from the elution step can be concentrated by flashevaporation. It will be within the scope of the skilled person in theart to determine suitable conditions for evaporation. Suitable exemplaryconditions for evaporation include:

-   -   feed flow rate: 150-200 L/hr; preferably about 180 L/hr    -   feed preheat temperature: 40-50° C.; preferably about 45° C.    -   recycle rate: 180-200 L/hr; preferably about 195 L/hr    -   operating vacuum: 5-10 kPa_(abs); preferably about 8 kPa_(abs)    -   vessel temperature: 30-40° C.; preferably about 32-36° C.

There is therefore provided a method for producing a molasses extractwith a high relative abundance of hydrophobic compounds includingpolyphenols at levels 5 to 10 fold higher than molasses, comprising thesteps of:

-   -   a. diluting the molasses to produce a 10 to 40% w/v aqueous        solution;    -   b. optionally filtering the diluted molasses produced in step        (a);    -   c. contacting the diluted molasses with a hydrophobic polymeric        adsorbent under conditions sufficient to enable binding of        compounds to the adsorbent and flow through of all other        compounds in the diluted molasses;    -   d. optionally passing the flow through from step (c) over the        hydrophobic polymeric adsorbent at least once;    -   e. optionally rising the hydrophobic polymeric adsorbent; and    -   f. eluting the compounds bound to the hydrophobic polymeric        adsorbent to produce the extract

wherein the extract produced in step (f) has a high relative abundanceof hydrophobic compounds including polyphenols levels 5 to 10 foldhigher than the sample of molasses prior to it being subjected to steps(b) to (f). Preferably the hydrophobic compounds are eluted with 30 to70% ethanol, most preferably 40% ethanol, and the molasses extractcontains hydrophobic polyphenols in an amount of at least at least 9000mg CE/100 g, more preferably at least 18000 mg CE/100 g, and mostpreferably at least 21000 mg CE/100 g.

This method produces a concentrated extract suitable for freeze drying.

Other than optionally filtering the molasses for the purpose of removingsediment and undissolved matter in step (b), this method of producing anextract does not subject the molasses to any pre-treatment steps toremove other substances prior to exposing the molasses to the polymericadsorbent. Prior art methods subject polyphenol containing substances toother purification and filtration steps to remove the higher molecularweight compounds and polyphenols, which impart undesirable colour andtaste to the substance. The method of the invention does not include anysteps for the specific purpose of removing these polyphenols or othercomponents, and the extract of the invention generated by hydrophobicpolymeric adsorption of molasses retains a number of these polyphenols.There is therefore provided a method for producing a molasses extractwith a high relative abundance of hydrophobic compounds includingpolyphenols at levels 5 to 10 fold higher than molasses, wherein themolasses is not subject to any pre-treatment steps prior to contactingthe molasses with the hydrophobic polymeric adsorbent that would removehigh molecular weight compounds including high molecular weightpolyphenols (other than in sediment or undissolved matter mentionedabove). Put another way, the molasses, or the diluted molasses used inthe method of the invention to produce a molasses extract with a highrelative abundance of hydrophobic compounds is untreated molasses, whichhas not been treated to remove high molecular weight compounds.

In a further embodiment of the invention, there is also provided anextract from molasses with a high relative abundance of hydrophobiccompounds including polyphenols when produced by a method of theinvention.

In an alternative method of producing an extract from molasses with ahigh relative abundance of hydrophobic compounds including polyphenols,the hydrophobic compounds may be removed from molasses using a selectiveabsorption process, whereby hydrophobic compounds, includingpolyphenols, are extracted using suitable solvents. Suitable solventsinclude but are not limited to ethanol, methanol, acetone and ethylacetate, or mixtures thereof, and dilutions thereof such as 50/50ethanol/water. The solvent containing the polyphenols may then besubjected to additional purification steps to remove any solid matter,prior to vacuum evaporation to produce the extract.

ii) Characterisation of Molasses Extract

The molasses extract of the invention contains hydrophobic polyphenolsin an amount of at least 9000 mg CE/100 g, more preferably at least18000 mg CE/100 g, and most preferably at least 21000 mg CE/100 g. Asexplained earlier, “CE”, or “catechin equivalent” is a measure of totalpolyphenolic content, expressed as mg catechin equivalents/g crudematerial.

The polyphenols in the molasses extract of the invention can bepolyphenols in free form or as a glycoside or can be a phenolic acid.For example, the molasses extract may include one or more of thefollowing polyphenols, polyphenol glycosides and phenolic acids:

-   -   free form polyphenols selected from one or more of apigenin,        catechin, catechin gallate, epicatechin, kaempherol, diosmin,        luteolin, quercetin, tricin, myricetin and diosmetin;    -   polyphenol glycosides selected from one or more of diosmin,        tricin-7-O-neohesperidoside, orientin, vitexin,        luteolin-8-C-(rhamnosylglucoside), schaftoside, isoschaftoside,        rutin; and    -   phenolic acids, selected from one or more of caffeic acid,        chlorogenic acid, p-coumaric acid, ferulic acid, gallic acid,        syringic acid, and vanillic acid.

Accordingly, in one embodiment of the invention, there is provided amolasses extract having a relatively high abundance of hydrophobiccompounds including polyphenols, wherein the polyphenols are present ina mixture of:

-   -   free form polyphenols selected from one or more of apigenin,        catechin, catechin gallate, epicatechin, kaempherol, diosmin,        luteolin, quercetin, tricin, myricetin and diosmetin;    -   polyphenol glycosides selected from one or more of diosmin,        tricin-7-O-neohesperidoside, orientin, vitexin,        luteolin-8-C-(rhamnosylglucoside), schaftoside, isoschaftoside,        rutin;    -   and phenolic acids, selected from one or more of caffeic acid,        chlorogenic acid, p-coumaric acid, ferulic acid, gallic acid,        syringic acid, and vanillic acid,        and are present in an amount of at least 9000 mg CE/100 g, or at        least 18000 mg CE/100 g, or at least 21000 mg CE/100 g.

Preferably, the free form polyphenols are at least catechin,epicatechin, and quercetin, the polyphenol glycoside is diosmin, and thephenolic acids are at least caffeic acid, chlorogenic acid, p-coumaricacid, ferulic acid, and syringic acid. More preferably, catechin ispresent in the amount of 150 to 200 mg/kg, epicatechin is present in theamount of 150 to 220 mg/kg, quercetin is present in the amount of 80 to150 mg/kg, diosmin is present in the amount of 410 to 425 mg/kg, caffeicacid is present in the amount of 100 to 320 mg/kg, chlorogenic acid ispresent in the amount of 100 to 400 mg/kg, p-coumaric acid is present inthe amount of 1100 to 1300 mg/kg, ferulic acid is present in the amountof 700 to 760 mg/kg, and syringic acid is present in the amount of 400to 500 mg/kg.

Polyphenol glycosides may be O linked or C linked glycosides.O-glycosides, such as diosmin, tricin, and rutin are hydrolysable, andare broken down either by bacterial enzymes in the intestine, or humanenzymes in the intestinal cell wall to unconjugated polyphenol aglyconeswhich are very easily absorbed. However, prior to absorption, theaglycones can be conjugated with glucuronic acid, such that very littleunconjugated polyphenol is actually absorbed.

The C-glycosides however, including those that lose the O-glycosidemoiety to expose the C-glycoside part of the molecule, have a C—C bondthat is not hydrolysable by enzymes or acids. C-glycosides thereforeremain intact, and without being bound to any theory, can behave assubstrates for the glucose transporters in the intestine and kidney,thereby blocking glucose transport. Orientin, vitexin,luteolin-8-C-(rhamnosylglucoside), schaftoside and isoschaftoside areall C-glycosides. Accordingly, in one embodiment of the invention, themolasses extract is enriched for C-glycosides, including but not limitedto orientin, vitexin, luteolin-8-C-(rhamnosylglucoside), schaftoside andisoschaftoside.

The molasses extract of the invention having a relatively high abundanceof hydrophobic compounds including polyphenols, in an amount of at leastof at least 9000 mg CE/100 g, or at least 18000 mg CE/100 g, or at least21000 mg CE/100 g, also contains trace elements, carbohydrates includingsmall amounts of sugars, moisture, ash and protein.

The molasses extract of the invention preferably contains the followingtrace elements, shown as element (mg) per weight of extract.

TABLE 1 Trace Element Concentration range Preferred concentrationCalcium 8000-9000 mg/kg 8800 mg/kg Iron  800-1000 mg/kg  860 mg/kgMagnesium 1500-2500 mg/kg 2000 mg/kg Manganese   50-100 mg/kg  65 mg/kgPotassium  100-250 mg/kg  190 mg/kg Sodium 10-50 mg/100 g 30 mg/100 g

The molasses extract of the invention also preferably contains no orminimal monosaccharide and disaccharide sugars. By “minimal sugars” itis meant that the total of fructose, glucose, sucrose, maltose, lactoseand maltotriose is less than 2 g per 100 g of extract, and preferablyless than lg per 100 g of extract as per the following:

TABLE 2 Sugars Concentration Fructose <0.2 g/100 g Glucose <0.2 g/100 gSucrose   0.3 g/100 g Maltose <0.2 g/100 g Lactose <0.2 g/100 gMaltotriose <0.2 g/100 g Total Sugars   <1 g/100 g

The molasses extract of the invention contains no detectable fat, orfatty acids present in an amount above 0.1 g/100 g of extract. Theextract is also low in moisture. By “low in moisture” it is meant thatthere is less than 10 g of moisture per 100 g extract, or less than 10%,and preferably less than 6 g/100 g extract, or less than 6%.

The molasses extract of the invention may also contain ash, in an amountof 1 to 5 g of ash per 100 g of extract, preferably 2.5 g to 3.5 g ofash per 100 g of extract.

By detecting nitrogen, it is possible to determine protein content ofthe molasses extract of the invention. In this regard, the proteincontent was estimated to be 5 to 20 g/100 g of extract, preferably 10 to15 g/100 g of extract and most preferably 12 to 13 g/100 g of extract.Other than protein, other N-containing compounds present in the extractmay be alkaloids.

Carbohydrates other than monosaccharides and sucrose are present in anamount of approximately 25 to 50 g/100 g of the extract, preferably 30to 40 g/100 g.

While the mono and disaccharide content of the molasses extract is lessthan 2 g per 100 g of extract, and preferably less than lg per 100 g ofextract, polymeric glycosyl residues are contained within the extractand form part of the carbohydrate content of the molasses extract as perthe following:

TABLE 3 Glycosyl residue: Mass/300 ug Mol %¹ Arabinose (Ara) 0.6 0.6Rhamnose (Rha) 0.8 0.8 Fucose (Fuc) n.d. — Xylose (Xyl) 0.9 0.9Glucuronic Acid (GlcA) n.d. — Galacturonic acid (GalA) n.d. — Mannose(Man) 0.2 0.1 Galactose (Gal) 0.1 0.1 Glucose (Glc) 112.2  97.5 N-Acetyl Galactosamine (GalNAc) n.d. — N-Acetyl Glucosamine (GlcNAc)n.d. — N-Acetyl Mannosamine (ManNAc) n.d. — TOTAL 114.7  ¹Values areexpressed as mole percent of total carbohydrate. The total percentagemay not add to exactly 100% due to rounding; n.d = not detected

The extract of the invention therefore preferably comprises about 25 to50 g/100 g of extract, preferably about 30 to 40 g/100 g, of which morethan 90% is polymers of glucose residues (not free glucose).

Accordingly, in one embodiment of the invention, there is provided amolasses extract with a high relative abundance of hydrophobic compoundsincluding polyphenols present in an amount of at least 9000 mg CE/100 g,or at least 18000 mg CE/100 g, or at least 21000 mg CE/100 g, andfurther comprising one or more of:

-   -   trace elements selected from one or more of calcium, iron,        magnesium, manganese, potassium and sodium;    -   protein and other nitrogen-containing compounds; and    -   carbohydrates other than monosaccharides and sucrose

wherein the extract has less than 2 g of monosaccharides and sucrose per100 g of extract.

More preferably there is provided a molasses extract with a highrelative abundance of hydrophobic compounds including polyphenolswherein the extract comprises

-   -   at least 9000 mg CE/100 g of hydrophobic polyphenols in a        mixture of free form polyphenols selected from apigenin,        catechin, catechin gallate, epicatechin, kaempherol, luteolin,        quercetin, tricin, myricetin and diosmetin; polyphenol        glycosides selected from diosmin, tricin-7-O-neohesperidoside,        orientin, vitexin, luteolin-8-C-(rhamnosylglucoside),        schaftoside, isoschaftoside, and rutin; and phenolic acids,        selected from caffeic acid, chlorogenic acid, p-coumaric acid,        ferulic acid, gallic acid, syringic acid and vanillic acid;    -   trace elements selected from one or more of calcium, iron,        magnesium, manganese, potassium and sodium;    -   protein and other nitrogen-containing compounds; and    -   carbohydrates other than monosaccharides and sucrose        wherein the extract has less than 2 g of monosaccharides and        sucrose per 100 g of extract.

Preferably, the hydrophobic polyphenols are present in an amount of atleast 18000 mg CE/100 g, or at least 21000 mg CE/100 g.

In each of the embodiments described above that contain trace elements,the trace elements that are present are present in the amounts of8000-9000 mg calcium/kg of extract, 800-1000 mg of iron/kg of extract,1500-2500 mg of magnesium/kg of extract, 50-100 mg of potassium/kg ofextract, 100-250 mg of potassium/kg extract and 10-50 mg sodium/100 g ofextract. In the embodiments that contain protein and N-containingcompounds such as alkaloids, the protein and N-containing compounds arepresent in an amount of 1 to 15 g of protein and N-containingcompounds/100 g of extract.

The carbohydrate component of each of the embodiments described above is25 g to 50 g/100 g of extract, preferably 30 to 40 g/100 g, and of that,more than 90% is polymeric glucose residues.

The molasses extract of the invention with a high relative abundance ofhydrophobic compounds including polyphenols has a high Oxygen RadicalAbsorbance Capacity (ORAC) value. The ORAC value was calculated by themethod described in Cao G, Alessio H, Cutler R (1993). “Oxygen-radicalabsorbance capacity assay for antioxidants”. Free Radic Biol Med 14 (3):303-11. Raw bran is reported to be one of the best antioxidant foodproducts with an ORAC value of 312400 μmol TE/100 g. The molassesextract of the invention, having a total ORAC value in the range of350000 to 385000 μmol TE/100 g, is at least 20% higher.

iii) Use of the Molasses Extract

As mentioned above, the inventors have found that administration of theextract of the invention can achieve important physiological effects andimportant health outcomes for the individual to which the extract isadministered. The extracts as described herein may be used in atherapeutic capacity in order to treat and/or prevent a number ofconditions.

Earlier work by the applicants showed that administration of a molassesfiltration extract to an animal was able to alter the distribution ofbody mass by increasing the proportion of lean mass to fat mass whencompared to the consumption of the same food without the addition ofthese compounds. These body mass altering compounds include polyphenolsand milk bioactives (WO2006/128259). In the current application, it hasbeen found that the molasses extract of the invention having arelatively high abundance of hydrophobic compounds including polyphenolscan reduce overall body fat and/or minimise fat accumulation byincreasing energy excretion and/or by influencing mechanisms involved infat and sugar oxidation and insulin sensitivity.

Accordingly, in one aspect of the invention, there is provided a methodfor decreasing body fat and/or minimise fat accumulation in an animal byadministering a composition including a molasses extract having arelatively high abundance of hydrophobic compounds including polyphenolsand a pharmaceutically acceptable carrier, excipient or diluent in anamount effective to decrease total body fat and/or minimise fataccumulation of the animal.

By “decreasing body fat”, it is meant that the animal has a decrease intheir amount of body fat. By “minimising fat accumulation” it is meantthat the animal does not increase its amount of body fat.

The phrase “in an amount effective” is used herein to refer to an amountwhich is sufficient to achieve the desired outcome. For example, anamount effective to decrease body fat, minimise fat accumulation or anamount effective to reduce energy absorption. An example of an effectiveamount for animals is 1 to 5% of the diet, preferably 2 to 4% of thediet. Assuming that a human normally consumes 1000 g of food per day andthe normal consumption of polyphenols is 1 g/day, the effective amountis likely to be in the range from 10 to 50 g/day, more preferably 20 to40 g/day.

Without being bound by any theory, it is believed that the compositionincluding the molasses extract having a relatively high abundance ofhydrophobic compounds including polyphenols reduces energy absorptionand/or alters fat metabolism. It may also influence energy expenditure.Energy expenditure is mainly a sum of internal heat produced andexternal work. In this context it is energy expenditure as a result ofinternal heat production. The internal heat produced is, in turn, mainlya sum of basal metabolic rate (BMR) and the thermic effect of food. Thecomposition including the molasses extract having a relatively highabundance of hydrophobic compounds including polyphenols may increasethe metabolism of the individual receiving the extract of the inventionie increase energy expenditure.

Accordingly, in another embodiment of the invention, there is provided amethod of reducing energy absorption and/or altering fat metabolism byadministering a composition including a molasses extract having arelatively high abundance of hydrophobic compounds including polyphenolsand a pharmaceutically acceptable carrier, excipient or diluent in anamount effective to reduce energy absorption and/or alter fatmetabolism.

In one embodiment of the invention, there is provided a method ofreducing energy absorption by administering a composition including amolasses extract having a relatively high abundance of hydrophobiccompounds including polyphenols and a pharmaceutically acceptablecarrier, excipient or diluent in an amount effective to reduce energyabsorption. More specifically the molasses extract increases theexcretion of, or decreases the absorption of, carbohydrates. Theinvention therefore provides a method of reducing energy absorptionand/or increasing energy excretion by administering a compositionincluding a molasses extract having a relatively high abundance ofhydrophobic compounds including polyphenols and a pharmaceuticallyacceptable carrier, excipient or diluent in an amount effective todecrease the absorption of carbohydrates and/or increase the excretionof carbohydrates.

In another embodiment of the invention, there is provided a method ofaltering fat metabolism by administering a composition including amolasses extract having a relatively high abundance of hydrophobiccompounds including polyphenols and a pharmaceutically acceptablecarrier, excipient or diluent in an amount effective to alter fatmetabolism.

In one embodiment of the invention, there is provided a method ofincreasing basal metabolic energy expenditure in a mammal, preferably ahuman, by administering a composition including a molasses extracthaving a relatively high abundance of hydrophobic compounds includingpolyphenols and a pharmaceutically acceptable carrier, excipient ordiluent in an amount effective to increase basal metabolic rate, therebyincreasing basal metabolic energy expenditure.

In preferred embodiments, the extract represents up to 1%, 2%, 3% and 4%of the diet.

Compositions containing extracts of the invention are also envisaged tobe able to improve fatigue and energy levels in healthy adults. In thisembodiment of the invention, there is provided a method of alleviatingor reducing the severity of fatigue by administering a compositionincluding a molasses extract having a relatively high abundance ofhydrophobic compounds including polyphenols and a pharmaceuticallyacceptable carrier, excipient or diluent in an amount effective toreduce energy absorption Similarly, it is believed that administrationof a composition including an extract of the invention can improve andelevate energy levels in an animal to which the composition isadministered in an effective amount.

In a preferred embodiment, the extract represents at least up to 1%, 2%,3% and 4% of the diet.

Consumption of the extracts of the invention is also thought to haveother beneficial effects on individuals who are overweight. While thereare a number of reasons why individuals are obese or overweight, it hasbeen suggested that those individuals may have a deficiency in theirsatiation response to sucrose (Linton et al. 1972). By satiationresponse, or satiety, it is meant the feeling of fullness orgratification following consumption of food (ie postprandial satiety).

There is therefore provided a method of improving postprandial satietyin an individual by administering a composition including a molassesextract having a relatively high abundance of hydrophobic compoundsincluding polyphenols in an amount effective to decrease a desire tohave further food.

As an alternative to administering a composition in the methods of theinvention, the individual may be administered the extract of theinvention as part of a satiety inducing food.

Administration of extracts of the invention has also demonstratedeffects on adipokine (gut hormone) levels, and as noted above, maytherefore also have an effect on fat metabolism. One such hormone isadiponectin, a protein hormone that modulates a number of metabolicprocesses, including glucose regulation and fatty acid catabolism.Despite being exclusively secreted from adipose tissue into thebloodstream levels of the hormone are inversely correlated with body fatpercentage in adults. Without being bound by any theory of action, it isbelieved that the extracts of the invention directly or indirectly leadto increased levels of adiponectin. As such, in another embodiment ofthe invention, there is provided a method of upregulating expression ofadiponectin by administering a composition including a molasses extracthaving a relatively high abundance of hydrophobic compounds includingpolyphenols and a pharmaceutically acceptable carrier, excipient ordiluent in an amount effective to upregulate expression of adiponectin.

Administration of extracts of the invention can also lead to upregulatedexpression of particular genes involved in regulation of energyexpenditure in the liver. For example, PPAR alpha and uncoupling protein2 (UCP2). PPAR-alpha is a regulator of lipid metabolism in the liver.Activation of PPAR-alpha promotes uptake, utilization, and catabolism offatty acids by upregulation of genes involved in fatty acid transportand peroxisomal and mitochondrial fatty acid (3-oxidation. UCP2 isthought to have a role in fatty acid oxidation and energy expenditurethrough thermogenesis.

Without being bound by any theory of action, it is believed that theextracts of the invention directly or indirectly lead to increasedlevels of genes including PPAR-alpha and UCP2. As such, in anotherembodiment of the invention, there is provided a method of upregulatingexpression of PPAR-alpha and/or UCP2 by administering a compositionincluding a molasses extract having a relatively high abundance ofhydrophobic compounds including polyphenols and a pharmaceuticallyacceptable carrier, excipient or diluent in an amount effective toupregulate expression of PPAR-alpha and/or UCP2.

By “upregulating expression” it is meant increased levels oftranscription of the gene, and optionally increased levels of expressionof the protein product. In a further embodiment, there is provided amethod of decreasing body fat and/or minimise fat accumulation in ananimal by administering a composition including a molasses extracthaving a relatively high abundance of hydrophobic compounds includingpolyphenols and a pharmaceutically acceptable carrier, excipient ordiluent in an amount effective to upregulate expression of one or moreof adiponectin, PPAR-alpha and UCP2.

In yet another embodiment there is provided a method of improvingpostprandial satiety in an individual by administering a compositionincluding a molasses extract having a relatively high abundance ofhydrophobic compounds including polyphenols and a pharmaceuticallyacceptable carrier, excipient or diluent in an amount effective toupregulate expression of one or more of adiponectin, PPAR-alpha andUCP2.

Administration of a composition of the invention may also be used inmethods for:

-   -   preventing and treating obesity, fatty liver, alcoholic liver,        diabetes and hyperlipidemia;    -   inhibiting absorption of saccharide in the body and weight        increase;    -   activating bifidus bacteria;    -   enhancing antioxidant activity;    -   improving insulin sensitivity/responsiveness;    -   enhancing hypoglycaemic activity;    -   enhancing tyrosine kinase inhibitory activity;    -   treating pre- and post-menstrual syndromes    -   treating cancer, headaches, dementia and alcoholism;    -   enhancing a-amylase inhibitory activity;    -   muscular strength enhancement;    -   activating mitochondria.

It may also exhibit neuroprotective effects.

There is also provided use of an effective amount of a molasses extracthaving a relatively high abundance of hydrophobic compounds includingpolyphenols in the preparation of medicament for decreasing body fat,minimising fat accumulation reducing energy absorption and/or alteringfat metabolism, increasing energy expenditure, improving and elevatingenergy levels in an animal, decreasing the absorption of carbohydratesand/or increasing the excretion of carbohydrates and improvingpostprandial satiety.

In a further embodiment, there is provided an effective amount of amolasses extract having a relatively high abundance of hydrophobiccompounds including polyphenols for decreasing body fat, minimising fataccumulation, reducing energy absorption and/or altering fat metabolism,improving and elevating energy levels in an animal, decreasing theabsorption of carbohydrates and/or increasing the excretion ofcarbohydrates and improving postprandial satiety.

The invention also provides a composition for decreasing body fat,minimising fat accumulation, reducing energy absorption and/or alteringfat metabolism, improving and elevating energy levels in an animal,decreasing the absorption of carbohydrates and/or increasing theexcretion of carbohydrates and improving postprandial satiety, thecomposition comprising as an active ingredient a molasses extract havinga relatively high abundance of hydrophobic compounds includingpolyphenols.

iv) Use of the Molasses Extract as an Ingredient

The molasses extract of the invention having a relatively high abundanceof hydrophobic compounds including polyphenols may also be administeredto an animal as an ingredient in a food or beverage. The extracts arepreferably produced by the method of the invention.

The therapeutic compositions of the invention may also be incorporatedinto foods, including pet foods.

The extracts of the present invention may be incorporated into foodproducts and beverages. The extracts may be impregnated, mixed,emulsified, sprayed or coated onto carriers such as cellulose,methylcellulose, dextrose, cyclodextrose, cyclodextrin, maltitol, fibreand fibre containing bioactives to improve delivery. Delivery may alsobe enhanced with a range of surfactants, lipids, complexes, solvents andco-solvent pharmaceutical delivery systems known in the pharmaceuticalart to improve bioavailability, absorption and efficacy.

As used herein, the term “food” or “food product” includes any edibleproduct for human or non-human consumption, such as but not limited toconfectioneries, supplements, snacks (sweet and savoury),cocoa-containing foods, flavours, beverages, dietary supplements andformulations including supplements used in animal health and nutrition.Additional ingredients desired in the resulting food product may beadded at any point in the process. In one embodiment of the invention,the extracts are in the form of syrups that can be used as substitutesfor regular glucose and high fructose corn syrups from wheat, corn,agave, stevia etc., as a lower Glycemic Index (GI) option.

The extracts of the present invention may be incorporated into foods,beverages and nutraceuticals, including, without limitation, thefollowing:

-   -   Dairy Products—such as cheeses, butter, milk and other milk or        dairy containing beverages, spreads and dairy mixes, ice cream        and yoghurt;    -   Fat-Based Products—such as margarines, spreads, mayonnaise,        shortenings, cooking and frying oils and dressings;    -   Cereal-Based Products—comprising grains (for example, bread and        pastas) whether these goods are cooked, baked or otherwise        processed;    -   Confectioneries—such as chocolate, candies, chewing gum,        desserts, non-dairy toppings, sorbets, icings and other        fillings;    -   Sports nutrition products including powders, pre-mixes, juices,        energy bars, isotonic drinks and gelatine, starch based or        pectin jellies;    -   Beverages—whether hot or cold (coffee, tea, cocoa, cereal,        chicory and other plant extract based beverages), alcoholic        beverages, carbonated, non-carbonated and lightly carbonated        beverages including colas and other soft drinks, powdered soft        drinks, fruit and vegetable juice drinks, dietary supplement,        breakfast beverages, instant pre-mixes and meal replacement        drinks; sport drinks, energy drinks, flavoured water drinks;    -   animal feeds including pet foods for companion animals such as        dogs, cats and horses;    -   Miscellaneous Products—including eggs and egg products,        processed foods such as soups, pre-prepared pastas.

Similarly, food grade ingredients such as soluble fiber (e.g.oligofructosaccharide), insoluble fiber (e.g. sugar cane fiber, oatbran), flour, starch, modified starch, gelatine, or other food,pharmaceutical or cosmetic ingredients impregnated with or containingthe extract according to the invention, can produce a unique foodingredient with enhanced levels of hydrophobic compounds includingpolyphenols.

The present invention includes food products comprising an extractaccording to the invention alone as the active ingredient or incombination with other active ingredients.

In one embodiment, there is provided a breakfast beverage including amolasses extract having a relatively high abundance of hydrophobiccompounds including polyphenols.

In another embodiment, there is provided a carbonated or low carbonatedbeverage including a molasses extract having a relatively high abundanceof hydrophobic compounds including polyphenols. Carbonated and lowcarbonated beverages are also known in the art as soft drinks.

In yet another embodiment, there is provided a satiety inducing foodincluding a molasses extract having a relatively high abundance ofhydrophobic compounds including polyphenols.

In yet another embodiment, there is provided a pet food including amolasses extract having a relatively high abundance of hydrophobiccompounds including polyphenols, wherein the pet food is preferably forcompanion animals including cats, dogs and horses.

v) Use of the Molasses Extract to Reduce GI

The molasses extract of the invention having a relatively high abundanceof hydrophobic compounds including polyphenols may be added othersubstances or ingredients to reduce the GI of that substance. By “reducethe GI” it is meant that the GI of the substance to which the extract isadded is lowered compared to the naturally occurring GI of thesubstance. It does not have to make the substance itself low GI (ieGI<55), although it may in fact do so depending on the substance.

In particular, the molasses extract having a relatively high abundanceof hydrophobic compounds including polyphenols may be used to reduce theGI of mono- and disaccharides.

Monosaccharides are the most basic units of biologically importantcarbohydrates. They are the simplest form of sugar and are usuallycolorless, water-soluble, crystalline solids. Examples ofmonosaccharides include glucose (dextrose), fructose (levulose),galactose, xylose, ribose, mannose, rhamnose and xylopyranose.

For example, glucose (dextrose) was described above as being a suitablecarrier for the extract for use in foods. In addition to that,impregnating, mixing, emulsifying, spraying or coating the molassesextract having a relatively high abundance of hydrophobic compoundsincluding polyphenols on to glucose can reduce the natural GI ofglucose. In turn, food prepared using glucose with a reduced GI also hasa reduced GI.

There is therefore provided a monosaccharide having a GI that is reducedfrom its naturally occurring GI, comprising a molasses extract having arelatively high abundance of hydrophobic compounds including polyphenolsadded to the monosaccharide. There is also provided a monosaccharidehaving a GI that is reduced from its naturally occurring GI, consistingessentially of a molasses extract having a relatively high abundance ofhydrophobic compounds including polyphenols added to the monosaccharide.By “consisting essentially of it is meant that there is only the extractand the monosaccharide. The presence of any other ingredients would onlybe in trace amounts, and would not be present in sufficient amounts tohave any effect on, or to counteract the GI lowering characteristics ofthe molasses extract on the monosaccharide.

There is also provided a method for reducing the GI of a monosaccharide,comprising the addition of a molasses extract having a relatively highabundance of hydrophobic compounds including polyphenols to themonosaccharide. Preferably the monosaccharide is glucose. The molassesextract can be impregnated, mixed, emulsified, sprayed or coated on tothe monosaccharide.

There is also provided a method of reducing the GI of a food byreplacing a monosaccharide present in or used in the food with amonosaccharide to which a molasses extract having a relatively highabundance of hydrophobic compounds including polyphenols has been added,and wherein the monosaccharide to which the extract is added has areduced GI compared to the monosaccharide being replaced.

Addition of a molasses extract having a relatively high abundance ofhydrophobic compounds including polyphenols to disaccharides can alsoreduce the GI of the disaccharide. Disaccharides are formed when twomonosaccharides undergo a condensation reaction. Like monosaccharides,disaccharides also dissolve in water, taste sweet and are called sugars.Table 1 provides list of disaccharides, including their monomericcomponents. There is therefore provided a disaccharide having a GI thatis reduced from its naturally occurring GI, comprising a molassesextract having a relatively high abundance of hydrophobic compoundsincluding polyphenols added to the disaccharide. In another embodiment,there is provided a disaccharide having a GI that is reduced from itsnaturally occurring GI, consisting essentially of a molasses extracthaving a relatively high abundance of hydrophobic compounds includingpolyphenols added to the monosaccharide. There is further provided witha method for reducing the GI of a disaccharide, comprising the additionof a molasses extract having a relatively high abundance of hydrophobiccompounds including polyphenols to the disaccharide. Preferably thedisaccharide is sucrose. The molasses extract may be impregnated, mixed,emulsified, sprayed or coated on to the disaccharide.

There is also provided a method of reducing the GI of a food byreplacing a disaccharide present in or used in the food with adisaccharide to which a molasses extract having a relatively highabundance of hydrophobic compounds including polyphenols has been added,and wherein the disaccharide to which the extract is added has a reducedGI compared to the disaccharide being replaced. Preferably thedisaccharide is sucrose.

TABLE 4 Disaccharide Units Bond Sucrose One glucose monomer and onefructose α(1→2)β monomer Lactulose One galactose monomer and onefructose β(1→4) monomer Lactose One galactose monomer and one glucoseβ(1→4) monomer Maltose two glucose monomers α(1→4) Trehalose two glucosemonomers α(1→1)α Cellobiose two glucose monomers β(1→4) Kojibiose twoglucose monomers α(1→2) Nigerose two glucose monomers α(1→3) Isomaltosetwo glucose monomers α(1→6) β,β-Trehalose two glucose monomers β(1→1)βα,β-Trehalose two glucose monomers α(1→1)β Sophorose two glucosemonomers β(1→2) Laminaribiose two glucose monomers β(1→3) Gentiobiosetwo glucose monomers β(1→6) Turanose a glucose monomer and a fructoseα(1→3) monomer Maltulose a glucose monomer and a fructose α(1→4) monomerPalatinose a glucose monomer and a fructose α(1→6) monomer Gentiobiulosea glucose monomer and a fructose β(1→6) monomer Mannobiose two mannosemonomers α(1→2), α(1→3), α(1→4), or α(1→6) Melibiose a galactose monomerand a glucose α(1→6) monomer Melibiulose a galactose monomer and afructose α(1→6) monomer Rutinose a rhamnose monomer and a glucose α(1→6)monomer Rutinulose a rhamnose monomer and a fructose β(1→6) monomerXylobiose two xylopyranose monomers β(1→4)

As outlined above, the molasses extracts of the invention can be used assubstitutes for regular glucose and high fructose corn syrups fromwheat, corn, agave, stevia etc., as a lower Glycemic Index (GI) option.Alternatively, in another embodiment of the invention, the molassesextracts having a relatively high abundance of hydrophobic compoundsincluding polyphenols can be used to reduce the GI of such sweeteners.Corn syrup (also known as glucose syrup) is a food syrup made from thestarch of maize. It contains varying amounts of glucose, maltose andhigher oligosaccharides. Enzymatic processing of corn syrup convertsglucose to fructose, creating high fructose corn syrup (HFCS). HFCS issweeter and more soluble than corn syrup, and in the United States inparticular, is a cheaper alternative to sucrose. Accordingly, HFCS hasreplaced sucrose in the food industry, and is commonly used in manyprocessed foods and beverages.

There is therefore provided a corn syrup, particularly high fructosecorn syrup (HFCS), having a GI that is reduced from its naturallyoccurring GI, consisting of a molasses extract having a relatively highabundance of hydrophobic compounds including polyphenols added to theHFCS, together with a method for reducing the GI of HFCS, comprising theaddition of a molasses extract having a relatively high abundance ofhydrophobic compounds including polyphenols to HFCS. Preferably themolasses extract is impregnated, mixed, emulsified, sprayed or coated onto the HFCS.

There is also provided a method of reducing the GI of a food byreplacing HFCS used in the food with HFCS to which a molasses extracthaving a relatively high abundance of hydrophobic compounds includingpolyphenols has been added, and wherein the HFCS to which the extract isadded has a reduced GI compared to the HFCS being replaced.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

The invention is now described with reference to the followingnon-limiting examples.

EXAMPLES

1. Molasses Extract Production Method

An extract from molasses was prepared by the method of the invention butsubject to a number of variables as per the following:

TABLE 5 Method A Step Method B Diluted to 20% molasses with waterMolasses preparation Diluted to 20% molasses with water CentrifugationMolasses clarification Filtration through a 0.1 μM stainless steelfilter Sample applied to column for at Hydrophobic adsorbent Samplecycled 3 times over column least about 60 minutes, at ambient columnpurification at a flow rate of 4 L/min temperature Purified water WashPurified water 70% Ethanol Elution 40% Ethanol

The total phenolics were measured in an extract derived from method Aand compared to method B. The amount of polyphenols in the molassesextract were determined as described in Kim, Dae-Ok, et al (2003)Antioxidant capacity of phenolic phytochemicals from various cultivarsof plums. Food Chemistry, 81, 321-26.

Method A batch 185.4 mg CE/g (18540 mg CE/100 g) Method B batch 193.3 mgCE/g (19330 mg CE/100 g)

Method B was found to have 5% more phenolic content, indicating anadvantage to filtering the extract, and eluting it with 40% ethanol. Thelower concentration ethanol solution is also advantageous from a costand safety perspective.

2. Characterisation of the Molasses Extract

i) Polyphenols

To further characterize the types of polyphenols found in the molassesextract of the invention, a combined HPLC and tandem mass spectrometryprocedure was performed.

Sample Extraction and Preparation: 1.5 g of extract was placed into 10mL of 80% methanol (150 mg/mL), and extracted overnight at 4° C. Afterextraction the sample was centrifuged at 18,000×g for 10 minutes topellet particulate matter. 1 mL of the centrifuged material was placedinto a glass tube, and the sample was dried completely using air. Oncedried the sample was resuspended in 500 μL of 0.1% formic acid(concentration ˜300 mg extract), for HPLC analysis.

HPLC Analysis: An Agilent 1100 HPLC with Diode Array Detector was usedfor all analyses. The HPLC Column used was a Phenomenex Luna 5u C18(2)100 angstrom 250×4.6 mm, with Widepore C18 4×2.0 mm guard cartridge. Allsolvents were of HPLC grade, and doubly distilled water was used.Solvent A: 2% acetonitrile/0.1% formic acid Solvent B: 90%acetonitrile/0.1% formic acid.

Column washed with 100% Solvent B, and equilibrated in 100% Solvent Aprior to the start of the run. 20 μL of the resuspended sample (˜15 mgextract) was injected onto the column. For the gradient andfractionation of the extract, the column was equilibrated at 0% SolventB followed by 0-30% Solvent B (2-27% acetonitrile) over 90 minutes; then30-100% Solvent B (27-90% acetonitrile) over 1 minute; then hold 100%Solvent B (90% acetonitrile) over 10 minutes; then 100-0% Solvent B(90-2% acetonitrile) over 1 minute; then hold 0% Solvent B (2%acetonitrile) for 8 minutes. Fractions were collected in 5 minuteintervals over the course of the HPLC run (signals monitored: 262 nm, 4nm bandwidth—reference 308 nm, 40 nm bandwidth), air dried, andsubjected to mass spectrometric (MS) analysis.

MS analysis identified a number of peaks identified as being thefollowing:

-   -   free form polyphenols—apigenin, catechin, catechin gallate,        epicatechin, kaempherol, diosmin, luteolin, quercetin , tricin,        myricetin and diosmetin    -   polyphenol glycosides—diosmin, tricin-7-O-neohesperidoside,        orientin, vitexin, luteolin-8-C-(rhamnosylglucoside),        schaftoside, isoschaftoside, and rutin    -   phenolic acids—caffeic acid, chlorogenic acid, p-coumaric acid,        ferulic acid, gallic acid, syringic acid and vanillic acid.

Quantification of Selected Polyphenols

The levels of a number of the polyphenols was determined in aqueousacidic and basic fractions of the molasses extract following ethylacetate extraction. Ethyl acetate extraction was carried out on bothacidified and basified aqueous solutions of the samples to ensure thatas many compounds as possible would be extracted from the samples. Analiquot of each sample (˜200 mg) was dissolved in water (10 mL) that hadbeen acidified (pH 1.6) or basified (pH 9.6). Methyl-4-formyl benzoate(7.4 μg) was added to each solution as an internal standard (ISTD). Themixtures were then extracted with ethyl acetate (2×10 mL), the solventwas evaporated under vacuum (40° C.) and the mixtures were reconstitutedin aqueous formic acid (0.1%, 5 mL) before subjected to HPLC and LC/MSanalysis.

TABLE 6 Amount in sample (mg/Kg) Extract - Acid Extract - Basic Apigenin34.8 not detected Catechin not detected 175.2 Epicatechin 168.8 207.4Luteolin 18.6 41.3 Quercetin 91.3 137.1 Rutin 59.3 59.3 Diosmin 303.6114.3 Caffeic Acid 168.8 207.4 Chlorogenic Acid 368.2 123.1 p-CoumaricAcid 1170.9 1253.8 Ferulic Acid 738.8 724.1 Syringic Acid 433.5 472.6Vanillic Acid 2.13 not detected

ii) Trace Elements

Trace elements were determined by inductively coupled plasma-massspectrometry (ICP-MS) and inductively coupled atomic emissionspectrometry (ICP-OES). 10 g of molasses extract was used for theanalysis. The sample was homogenised and a sub-sample (0.2-0.5 g)digested with re-distilled nitric acid on a DigiPrep block for one houruntil vigorous reaction was complete. Samples were then transferred to aMilestone microwave to be further digested. After making up toappropriate volume with Milli-Q (high purity) water, the digest wasanalysed for trace elements using ICP-MS and / or ICP-OES. The resultswere as follows:

TABLE 7 Trace Elements Concentration Calcium 8800 mg/kg Iron  860 mg/kgMagnesium 2000 mg/kg Manganese  65 mg/kg Potassium  190 mg/kg Sodium 30mg/100 g

iii) Sugar Determination

About 15 grams of molasses extract was used for the analysis. The samplewas homogenised and a sub sample was accurately weighed. Sugars wereextracted with 25 mL water at 60° C. for 30 minutes. The extract wasclarified with 25 ml acetonitrile and filtered through a 0.45 um filterinto a 2 mL vial, suitable for HPLC. For the determination of commonsugars, the filtered solution was analysed by HPLC using amino columnwith an acetonitrile/water mobile phase containing salt and refractiveindex detection. Quantitation was made against a standard solutioncontaining known amounts of fructose, glucose, sucrose, maltose andlactose. For the determination of low level sugars, the filteredsolution was analysed by HPLC using carbohydrate ES column with anacetonitrile/water mobile phase and evaporative light scatteringdetector (ELSD). Quantitation was made against a standard solutioncontaining known amounts of fructose, glucose, sucrose, maltose andlactose. Result calculation was performed by the HPLC software and areport generated. The molasses extract contained the following sugars assummarized in the table below.

TABLE 8 Sugars Concentration Fructose <0.2 g/100 g Glucose <0.2 g/100 gSucrose   0.3 g/100 g Maltose <0.2 g/100 g Lactose <0.2 g/100 gMaltotriose <0.2 g/100 g Total Sugars   <1 g/100 g

iv) Protein

The total nitrogen was determined by the Kjeldahl method, as would beknown to the person skilled in the art, and the protein amount estimatedfrom that by multiplying the total nitrogen by a factor (6.25). Themolasses extract was homogenised and a sub sample (approx. 2 g) wasaccurately weighed into a Kjeldahl digestion tube. A digestion aid ofpotassium sulphate and a catalyst, copper sulphate was added to thesample, followed by 20 mL of concentrated sulphuric acid. The tube wasslowly heated to 400° C. and the temperature maintained until themixture in the tube was clear. The clear solution was digested for 1hour and the tube allowed to cool.

Once the tube had cooled 50 mL of distilled water was added. The tubewas placed in a Kjeltec distillation unit and the mixture was steamdistilled into a beaker containing 50 mL of saturated boric acidsolution. The distilled solution was titrated with standardised 0.1Nsulphuric acid solution using a mixed indicator of bromocresol green andmethyl red. Calculations: The following equation was used to calculatetotal nitrogen: Total N (g/100 g)=0.14*(titre-blank)/sample mass orvolume. For protein estimation the total N was multiplied by 6.25.

From this method, the protein content of the molasses extract wascalculated to be 12.6 g/100 g.

To identify the protein components of the molasses extract, a sample ofthe extract was run on an SDS-PAGE gel for visualization and massspectrometric analysis after in-gel trypsin digestion. 20 mg of molassesextract was dissolved in 1 mL of 1×XT Sample Buffer. The sample wascentrifuged at 16,000xg for 5 minutes to pellet any particulates.

Two SDS-PAGE gels were run, one for in-gel trypsin digestion and one forvisible protein analysis. 12% Criterion Bis-Tris XT gels were used, withtheir corresponding buffers. 30 μg of protein were run four times oneach gel.

In-Gel Digestion: Samples were loaded onto the gel in a total volume of26.25 μL (containing the 30 μg of protein). The gel was run at 75 V for5 minutes, and then 120 V for 10 minutes, which placed the proteins 1.5cm into the gel. The gel was fixed in 30% methanol/10% acetic acid for10 minutes, and washed twice with ddH₂O for five minutes each. Afterwashing, each of the four gel bands were cut to just below the dyefront, diced, and placed into 1.5 mL micro-centrifuge tubes to which 50mM ammonium bicarbonate/50% acetonitrile was added to destain thesamples.

After de-staining the samples were reduced with 30 mM dithiothreitol,and alkylated with 55 mM iodoacetamide. After alkylation the gel bandswere dehydrated, and then overlaid with a 1:20 ratio of trypsin toprotein, and covered in 100 mM ammonium bicarbonate to increase thesample pH to between 7-8. The bands were then allowed to digestovernight. Peptides were extracted using 50% acetonitrile/5% formicacid. After extraction the liquid containing peptides was dried,resuspended in 0.1% formic acid, and desalted using a ZipTip procedure.De salted samples were again dried and resuspended in 0.1% formic acidfor mass spectrometric analysis.

Tandem mass spectra were obtained and some peptide sequences werededuced by manual inspection. These peptide sequences were unique andnot found in any known genomic databases. In the absence of thesugarcane genome sequence it was not possible to confirm whether thesepeptide sequences are from sugarcane.

v) Carbohydrate Analysis

To get an empirical and more quantitative determination of thecarbohydrate content of the molasses extract the glycosyl composition ofthe extract was analysed. Glycosyl composition analysis was performed bycombined gas chromatography/mass spectrometry (GC/MS) of theper-O-trimethylsilyl (TMS) derivatives of the monosaccharide methylglycosides produced from a sample of the molasses extract by acidicmethanolysis.

20 μg of inositol was added to 300 μg of molasses extract. Methylglycosides were then prepared from the dry sample by methanolysis in 1MHCl in methanol at 80° C. (17 hours), followed by re-N-acetylation withpyridine and acetic anhydride in methanol (for detection of aminosugars). The sample was then per-O-trimethylsilylated by treatment withTri-Sil (Pierce) at 80° C. (0.5 hours). These procedures were carriedout as previously described in Merkle and Poppe (1994) Methods Enzymol.230: 1-15; York, et al. (1985) Methods Enzymol. 118:3-40. GC/MS analysisof the TMS methyl glycosides was performed on an Agilent 6890N GCinterfaced to a 5975B MSD, using a Supelco EC-1 fused silica capillarycolumn (30m x 0.25 mm ID).

TABLE 9 Glycosyl residue Mass (ug) Mol %¹ Arabinose (Ara) 0.6 0.6Rhamnose (Rha) 0.8 0.8 Fucose (Fuc) n.d. — Xylose (Xyl) 0.9 0.9Glucuronic Acid (GlcA) n.d. — Galacturonic acid (GalA) n.d. — Mannose(Man) 0.2 0.1 Galactose (Gal) 0.1 0.1 Glucose (Glc) 112.2  97.5 N-Acetyl Galactosamine (GalNAc) n.d. — N-Acetyl Glucosamine (GlcNAc)n.d. — N-Acetyl Mannosamine (ManNAc) n.d. — Total 114.7  ¹Values areexpressed as mole percent of total carbohydrate. The total percentagemay not add to exactly 100% due to rounding. “n.d” is not detectable.

As can be seen from the results in the table above, the carbohydratecontent of the molasses extract is predominantly composed of glucoseresidues with a small amount of other monosaccharides also detected.

vi) Moisture Content

About 10 g of molasses extract was homogenised for moisturedetermination by either using sand and vacuum drying (Sand method) or nosand and conventional drying (no sand method).

Sand method: A moisture dish with sand, lid and glass rod was oven-driedat 102° C. and cooled before all dried components were weighed togetherto the nearest 0.1 mg. 2 to 5 g of sample was weighed, to nearest 0.1mg, into the moisture dish. Water was added to the dish to aid mixing ofthe sample and sand. The moisture dish was placed on a steam bath untilvisible dryness of the sand/sample mix was achieved. The dish andcomponents were placed in a vacuum oven and dried under vacuum (approx.5 kpa) at between 70 and 100° C. depending on the sugar content of thesample. Drying time was a minimum of 4 hours depending on the samplematrix. After the required initial drying period the moisture dish andcomponents were removed, cooled, re-weighed and returned for a further 1hour drying. The weighing and drying process was repeated until constantweight is obtained.

No sand method: A moisture dish and lid was placed in the oven at 102°C. dried and cooled. The dried components were weighed together to thenearest 0.1 mg. A portion of sample (2 to 5 g) was weighed, to nearest0.1 mg, into the dish. The sample in the dish was then placed in aconventional oven at 102° C. for a minimum of 4 hours depending on thesample matrix. The dish and lid were then removed, cooled, re-weighedand returned for a further 1 hour drying. The weighing and dryingprocess was repeated until a constant weight was obtained.

To determine moisture content in both methods, the mass of the dish(plus components or lid depending on the method) was subtracted from themass of dried sample and dish (plus components or lid), then divided bythe sample mass obtained. The final result was then multiplied by 100 toobtain a result as % moisture or g/100 g. Both methods determined themoisture content of the molasses extract to be 5.9 g/100 g of extract.

vii) Ash Analysis

Ash content was determined by weighing 10 g of sample into a preparedweighed dish, beaker or crucible. The sample was dispersed on bottom ofcontainer, and excess moisture removed in a water bath. The containerwas then transferred to a muffle furnace and slowly heated to 525°C.±25° C. until all organic matter was destroyed. Dissolving salts inwater enhanced destruction of occluded carbon particles. The remainingash product was weighed and was found to be present as 3.1 g per 100 gof extract.

vii) Fat and Fatty Acid Analysis

Fat content was determined by the Mojonnier extraction method (Mills BLet al (1983) J Assoc Off Anal Chem 66(4):1048-50)). About 10 g ofmolasses extract was homogenised and a sub sample (approx. 2 g) wasaccurately weighed into a beaker. 10 mL of approx. 10% hydrochloric acidwas added and the mixture was heated at 80° C. until hydrolysis wascomplete (approx. 0.5 hours). The mixture was cooled and transferredquantitatively to a Mojonnier tube. 10 mL of ethanol was added and thefat was extracted by shaking for 1 minute with 25 ml of diethyl etherand a further minute with each of 25 ml of petroleum ether and 50 mlpetroleum and diethyl ether mix (The petroleum and diethyl ether mixextract was conducted twice). After each solvent addition, andsubsequent shaking, the organic layer was decanted from the Mojonniertube into a pre-weighed glass dish. Once all extractions were completethe organic extract in the glass dish was evaporated. The dish was thendried in an oven at 102° C. until constant weight was achieved.

Calculation: % Fat=[(Weight of dish−Weight of dish)/Weight ofsample]×100. Using this calculation, no detectable fat was found in themolasses extract of the invention, wherein “no detectable fat” was<0.2 gof fat/100 g of extract.

In addition to analysing the fat content of the molasses extract, fattyacid composition was also investigated, based on 10 g of molassesextract. The sample was homogenised and a sub sample taken (about 1 g).Fat was extracted from the sample using either Chloroform/Methanol orPetroleum ether/iso-propyl alcohol. The extract was evaporated undernitrogen. A minimum extracted mass of 0.2 g fat was required. Theextracted fat was esterified using a methanolic sodium methoxidesolution and treatment with sulphuric acid in methanol. The solution wasneutralised and re-extracted using n-hexane. The hexane layer wasremoved, dried using anhydrous sodium sulphate and made to volume, withhexane.

The relative proportion of each fatty acid methyl ester in the preparedsample was determined using gas chromatography with flame ionisationdetection. Identification of the individual fatty acids was made byretention time against a standard of known fatty acid methyl estersincluding both cis and trans isomers. The amount of Conjugated LinoleicAcid (CLA) can be also determined from the FAME's chromatogram.Instrument software was used to provide the calculation of proportionalmethyl ester concentrations.

As can be seen from the table below, no detectable fatty acids werepresent in the molasses extract (reported as g of fat/100 g of extract):

TABLE 10 Saturated fat <0.1 g/100 g Mono trans fat <0.1 g/100 gMono-unsaturated fat <0.1 g/100 g Omega 3 fats <0.1 g/100 g Omega 6 fats<0.1 g/100 g Poly trans fats <0.1 g/100 g Poly-unsaturated fat <0.1g/100 g Trans fats <0.1 g/100 g

viii) Antioxidant Activity

The ORAC value, indicative of the amount of antioxidant scavengingactivity of the molasses extract of the invention was calculated by themethod described in Cao G, Alessio H, Cutler R (1993). “Oxygen-radicalabsorbance capacity assay for antioxidants”. Free Radic Biol Med 14 (3):303-11. The ORAC Vitamin E equivalents of the extract were calculated tobe 383070 μmol TE/100 g.

3. Impact of Consumption of the Molasses Extract on Weight

In this example, an extract of the invention (prepared by Method Areferred to above in Example 1) was mixed into a high-fat,high-carbohydrate rodent diet to determine if their intake assists inpreventing the development of obesity, and that the extract does so byincreasing energy excretion and/or by influencing mechanisms involved infat and sugar oxidation and insulin sensitivity. The results describedbelow were also confirmed in a cat model (data not shown), which showedboth decreased body fat and minimisation of fat accumulation.

Methods

45 C57B1/6J male mice were maintained on a high fat-high carbohydratediet containing 2% or 4% of an extract of the invention or a controladditive. The animals were fed the diets for 12 weeks from six weeks ofage. During this 12 week period, the mice were monitored daily, theirfood and water intake and body weight were measured three times perweek, and faeces were collected to determine energy content.

While the animals were maintained on the experimental diets thefollowing procedures were performed:

Week 1—indirect calorimetry

Week 8—faecal energy excretion

Week 10—glucose tolerance test

Week 12—body composition (DEXA) was analysed and while still underanaesthetic the animals were killed by terminal bleeding (via cardiacpuncture) and blood and tissue (brain, adipose tissue, liver and muscle)samples were collected.

1. LabMaster—calorimetry and activity

In their first week on the experimental diets, mice were assessed in theLabMaster System. Prior to commencement of calorimetry measurement, theLabMaster system was calibrated with standard gases, drinkers and foodcontainers were filled and bedding was placed into the cages. Mice wereplaced into the LabMaster cages individually and allowed to acclimatefor 24 hours. Following this, the system recorded data for the animalfor a further 24 hours before the mouse was returned to its home cage.

2. Faecal energy excretion

Faeces samples (from week eight) were collected from each of the mice,and were placed, in individual foil containers in an oven at 83° C. for48 hours. Following this, each sample was ground into a powder using ahomogeniser and was pressed into a pellet (˜0.6 g) using a pellet press(Model 2811, Parr Instruments, Moline Ill.). The pellet was placed intoa crucible atop a support stand, and a 10 cm length of fuse wire wasfastened between the two electrodes. The sample was then arranged insidea bomb (Model 1108 oxygen bomb, Parr Instruments, Moline Ill.), with 1ml of water, which was flushed of atmospheric nitrogen and refilled withoxygen. Prior to commencement of the bombing procedure, the calorimeterwas calibrated using a benzoic acid standard in order to verify thechemistry of the combustion method and the precision of the energyamendments involved in the analysis of the results. The calorimeter(Model 1261 Parr Instruments, Moline, IL) was filled with two litres ofdeionised water, and the bomb was gently lowered inside, ensuring thatprior to submersion the ignition wires were inserted into the twoterminal sockets on the bomb head. The oxygen was then combusted, andthe pellet was ignited by the passage of current through the fuse wire.The temperature measurement took place directly in the bomb and caloricvalue was calculated from the heat released during the combustionprocess. This energy value of the faeces was calculated in MJ/kg.

3. Faecal Lipid, Carbon and Nitrogen Analysis

Faecal lipid content was determined by mixing 100 mg of ground faeceswith 4 ml of chloroform/methanol (2:1) and incubating at 60° C. for 30minutes. The samples were passed through a Whatman No.1 filter(Sigma-Aldrich Pty. Ltd., Castle Hill, NSW, Australia) into pre-weighedweighing boats and placed under a fume hood to allow solventevaporation. After the weight stabilised, the difference in weightbetween empty weighing boats and weighing boats containing the driedmaterial was the faecal lipid amount, which was expressed as apercentage of the weight of the starting faecal sample.

Carbon (C) and Nitrogen (N) analysis was carried out from driedhomogenised faeces powder. The faecal C and N contents were analysedusing the vario EL III CHNOS elemental analyser (ElementarAnalysensysteme GmbH, DonaustraBe, Germany) Approximately 20 mghomogenized samples were packed in tin foil and weighed. The sampleswere combusted and CO₂ was retarded in an adsorption trap. N₂ was thenmeasured directly in the thermal conductivity detector. After theN-measurement, the CO₂ was thermally desorbed and measured.

4. Glucose Tolerance Test

To assess glucose tolerance, mice were fasted overnight (with ad libwater). The mouse was placed into a restraint tube, and a basal fastingblood glucose level was obtained by removing the tip of the tail with arazor blade (approx.1 mm) and withdrawing ˜5 μl of whole blood into aheparin-containing microcuvette (Hemocue, Medipac Scientific NSW). Thiswas then inserted into the glucose monitor (Hemocue 201+, MedipacScientific, NSW) and the fasting glucose concentration was recorded.

Subsequently, the mice were injected intraperitoneally with glucosesolution (1 g/kg body weight) using a 0.5 ml diabetic syringe. Animalswere then returned to the home cage and additional blood samples wereobtained from the same tail cut at 30, 60 and 120 minutes post glucoseloading.

5. Body Composition Analysis by Dual Energy X-Ray Absorptiometry (DEXA)

Following 12 weeks on the experimental diets, in vivo body compositionof the mice was assessed using Dual energy x-ray absorptiometry (DEXA)(Norland pDEXA Sabre, Norland Medical Systems, White Plains, N.Y.).

Prior to scanning, the DEXA machine was calibrated using qualityassurance and quality control standards of known mass supplied by themanufacturer. The mice were anaesthetised by means of an injection intothe intraperitoneal cavity (ketamine 61 mg/kg and xylazine 9 mg/kg.),and were placed in the prone position on the DEXA scanning platform,with the tail secured by tape.

The animals were scanned and results were obtained for fat and fat-freemass, as well as bone mineral content and density. Once scanning wascomplete, the animals were killed by terminal bleeding (via cardiacpuncture) whilst still anaesthetised.

6. Enzyme-linked Immunosorbent Assays (ELISA)

The concentration of leptin and adiponectin present in the mice plasmawas quantified by Enzyme-linked immunosorbent assay (ELISA) (LINCO,Missouri USA). The ELISA microtiter plate wells came with antibodiesbound to the surface, and the antigen-containing sample was added tothis. The plate was then washed to remove the unbound proteins. Theantigen-specific antibodies were added, followed by a substrate designedto create an oxidative reaction with the enzyme labelled antibody andgenerate a colour formation proportionate to the amount of antigenpresent in the sample. Stop solution was then added to acidify thesample and cease the reaction. The enzyme activity was analysed in aspectrophotometer. The degree of absorbency detected by thespectrophotometer is directly proportionate to the amount of antigenpresent, the concentration was then elucidated from a reference curveproduced within the same assay from reference standards of knownconcentration.

7. In Vivo Visualization of Distribution of Adipose Tissue by MagneticResonance Imaging (MRI)

After 14 weeks (2 weeks after cessation of diet supplementation), thebody fat deposition of 6 mice (n=2 per group) was assessed with magneticresonance imaging (MRI). The animals were sacrificed using CO₂ gas,positioned prone on cardboard with their limbs splayed, placed on ice,and transported to the Howard Florey Institute at Melbourne University.

Regional body fat distribution was visualized by magnetic resonanceimaging (MRI). Images were acquired on a Bruker BIOSPEC 47/30 MRIscanner, equipped with a horizontal 4.7 Telsa Oxford magnet. Protondensity weighted axial images with the following parameters: number ofslices, 20; slice thickness, 1 mm; field of view (FOV), 6 cm; matrixsize, 256×256; repetition time (TR), 815 ms; echo time (TE), 17.9 mswere acquired.

8. Analysis of mRNA Expression

Total RNA was extracted from ˜100 mg of adipose or liver tissue usingTri-reagent (PE Applied Biosystems, CA, USA). Nanodrop 1000 (ThermoFisher Scientific Inc, MA, USA) was used to determine the purity of RNAand the ratio (A260/A280) values were close to 2.0. High capacity cDNAreverse transcription kit (PE Applied Biosystems, CA, USA) was used tosynthesise cDNA from 0.5 μg of RNA from the tissue in a total of 20 μLof reaction volume. Reverse transcription was performed by incubatingthe samples at 25° C. for 10 min, 37° C. for 120 min, 85° C. for 5secfollowed by 4° C. for 30 sec. RT-PCR amplification was performed using 1μ1 of cDNA diluted at 1:10 using gene specific primer sets (GeneWorksPty Ltd, SA, Australia). The oligonucleotide sequences of the forward(sense) and reverse (antisense) primers used for amplification were asin Table 1. Each primer set was used at a concentration of 3.75 μM in afinal volume of 25 μL using the Brilliant® II SYBR® Green QRT-PCR MasterMix Kit, 1-Step (Agilent Technologies, Inc., CA, USA). Real-time PCR wasperformed using the MX3000P qPCR machine (Agilent Technologies, Inc.,CA, USA) where target expression was normalised to the amount ofendogenous control (beta actin) relative to CON value, given by AACTmethod.

TABLE 11 Reverse Primer NCBI Forward Primer Accession Gene (5′-3′)(5′-3′) Number Beta actin CTATGCTCTCCCTCACGCCATC CCACGCTCGGTCAGGATCTTCNM_007393.3 (SEQ ID NO: 1) (SEQ ID NO: 2) AdiponectinGCCGCTTATGTGTATCGCTCAG GCCAGTGCTGCCGTCATAATG NM_009605.4 (SEQ ID NO: 3)(SEQ ID NO: 4) PPARγ2 GGAAGCCCTTTGGTGACTTTATGG GCAGCAGGTTGTCTTGGATGTCNM_011146.3 (SEQ ID NO: 5) (SEQ ID NO: 6) UCP2 GCTGGTGGTGGTCGGAGATACCATTACGGGCAACATTGGGAGAAG NM_011671.4 (SEQ ID NO: 7) (SEQ ID NO: 8) FASGGTTCTAGCCAGCAGAGTCTACAG CTCGTTGTCACATCAGCCACTTG NM_007988.3(SEQ ID NO: 9) (SEQ ID NO: 10)

9. Statistical Analysis

A two-way analysis of variance (ANOVA), with repeated measures on onefactor, (Statistica V7, Statsoft USA) was used to analyse body weightand food and water consumption between each group. All other data wereanalysed using one-way ANOVA. Sigmaplot (9.0, California, USA) wasutilised to calculate the area under the curve of the glucose tolerancetest. This was conducted using the trapezoidal rule and was followed bya one-way ANOVA to assess group differences. Post-hoc Fisher PLSD testswere conducted where appropriate. All results are presented as mean±SEM.A p- value of less than 0.05 was considered significant.

Results—Summary

Consumption by animals of the extract of the invention in theexperimental diet-induced-obesity model resulted in a >20% reduction ofbody fat (visceral and peripheral) and a concomitant decrease in overallbody weight of about 9% in a dose-dependent manner (statisticallysignificant) over the 12 week study period. Decreased body weight gainand body fat was observed after treatment with a 4% extract of theinvention. A consistent trend was observed after treatment with a 2%extract, but the results were not statistically significant for allparameters tested.

Detailed Results

Food and fluid intake: ANOVA analysis of the daily food and fluid intakepatterns of the mice throughout the experimental period confirmed thatthere was no difference in food intake between the groups at any timeduring the experiment (data not shown). This confirms that the decreasedbody weight and body fat was not attributable to decreased food intake.The 4% extract group however did drink more than the control group (datanot shown).

Fat levels: Dual Energy X-ray Absorptiometry (DEXA) was conducted at theend of the experimental period. The fat and fat-free mass of the micewas quantified and the results are illustrated in FIGS. 1A and B. ANOVAanalyses revealed that the 4% group had less fat mass than the controlgroup, F(2, 38)=3.32, p=0.047, however there were no differences betweenthe groups for the measure of fat-free mass, F(2, 38)=0.073, p=0.930.

Final body weight (determined by measuring the animals post-mortem) andbody weight measured by DEXA (which does not measure fluids) areillustrated in FIGS. 2A and B. They both demonstrate that the 4% grouphad lower body weight relative to control at the conclusion of theexperiment (F(2, 38)=3.54, p=0.040; F(2, 38)=3.94, p=0.030).

Adverse effects: Bone mineral density and bone mineral content were alsomeasured using DEXA. The results of these analyses indicated that therewas no difference between the groups for either of these measures (datanot shown), indicating that the extracts had no deleterious effects onbone growth or development over the 12 week study. The complete absenceof any acute or chronic toxicity or observable adverse physiological ormetabolic effects throughout the study period suggests that relativelyhigh doses of the extract of the invention are well tolerated in thispre-clinical animal model.

Glucose tolerance: Blood glucose levels were examined in the mice priorto and at 30-minute intervals following a glucose load. The results ofthe glucose tolerance test are shown in FIG. 3, and demonstrate thatthere were no differences between the three groups in their clearance ofglucose, F(2, 39)=0.59, p=0.558. The similarity in glucose-toleranceprofiles of between the animals consuming extracts of the invention andthose consuming a control diet confirms adequate pancreatic function inboth animal populations (i.e. no evidence of insulin-tolerancedeveloping).

Energy expenditure: In the first week of animals receiving theexperimental diets metabolic rate was measured via indirect calorimetryin the Labmaster system. At this time general locomotor activity wasalso measured. No differences were observed in either of these measures.No changes were observed in energy expenditure as evaluated by thesemethods, indicating that an increase in metabolism with extracts of theinvention may not be the mechanism of reduced weight gain (FIGS. 4A andB).

Faecal energy excretion: Excreted energy was assessed in the faecaloutput of the mice after 8 weeks on the experimental diets. The totalenergy output was greater in 4% extract treated mice compared to control(FIG. 5A). Digestibility of the energy within the diets was reduced inboth groups receiving extracts of the invention (FIG. 5B). Increasedfaecal energy excretion supports the hypothesis that that reduced energyabsorption is the mechanism responsible for reduced weight gain in theanimals consuming extracts of the invention.

Faecal Lipid, Carbon (C) and Nitrogen (N) content: The cause for theincrease in faecal energy mentioned above was examined There was nosignificant difference in the faecal lipid contents between theexperimental groups and the control group (FIG. 5C). One-way ANOVAanalyses revealed that the faecal C content, indicative of carbohydratelevel, in both PME treatment groups was higher relative to the controlgroup (FIG. 5D). The faecal N content, indicative of protein content,was lower in mice whose diet was supplemented with 4% PME when comparedto that of the control (FIG. 5E). The C/N ratio was significantlydifferent between the control mice and those whose diet was supplementedwith 4% PME (FIG. 5F).

Adipokines: The ELISA assay of plasma leptin identified significantlylower leptin levels in mice in the 4% group relative to the controlgroup (FIG. 6). Leptin is a 16 kDa protein hormone that plays a key rolein regulating energy intake and energy expenditure, including appetiteand metabolism. Leptin acts on receptors in the hypothalamus of thebrain where it inhibits appetite by counteracting the effects of feedingstimulants such as neuropeptide Y and anandamide, as well as promotingthe synthesis of a-MSH, an appetite suppressant. The absence of leptin(or its receptor) is thought to lead to uncontrolled food intake. Thefact that mice in the 4% group having decreased body fat also had lowerlevels of leptin substantiates the data relating to decreased body fataccumulation.

There were only minor differences in circulating plasma adiponectinlevels between any of the groups, suggesting that the reduced fatdeposition in animals receiving extracts of the invention was not due toincreased energy expenditure, but more likely reduced caloric adsorption(ie reduced energy absorption).

Gene expression—adipose tissue: Gene expression was analysed in theadipose tissue of animals from the experimental groups. Administrationof both 2% and 4% of the extract of the invention resulted in increasedexpression of adiponectin (released primarily by small adipocytes,involved in glucose regulation and fatty acid catabolism) and peroxisomeproliferator-activated receptor (PPAR) gamma (involved in fatty acidstorage and glucose metabolism) genes relative to control animals (FIG.7A and B). There were no differences in uncoupling protein 2 (UCP2)(involved in energy expenditure) (FIG. 7C). Administration of theextract of the invention at 4% also increased fatty acid synthase (FAS)expression, involved in the synthesis of fatty acids (FIG. 7D).Increases in gene expression of adiponectin, PPAR-gamma and FAS areconsistent with a reduction in body adiposity, possibly due to reducedenergy absorption. Increased expression of PPAR-gamma and FAS isconsistent with improved insulin sensitivity.

Liver mRNA expression: Gene expression was also analysed in the liver.Administration of both 2% and 4% of the extract of the inventiontreatment resulted in increased expression of PPAR alpha relative tocontrol animals (FIG. 8A) and UCP2 (FIG. 8B).

MRI analysis of the fat distribution: After 14 weeks (2 weeks after thecessation of dietary supplementation), the body fat deposition of 6 mice(n=2 per group) was assessed with magnetic resonance imaging (MRI).Consumption by animals of the extract of the invention in theexperimental diet-induced-obesity model resulted in a decrease in totalbody fat (visceral and peripheral). FIG. 9 illustrates a typical resultseen between control mice and those receiving 2% and 4% of the extractof the invention.

Modifications and improvements to the invention will be readily apparentto those skilled in the art. Such modifications and improvements areintended to be within the scope of this invention. The foregoingdescription details certain embodiments of the invention. It will beappreciated, however, that no matter how detailed the foregoing appearsin text, the invention can be practiced in many ways. As is also statedabove, it should be noted that the use of particular terminology whendescribing certain features or aspects of the invention should not betaken to imply that the terminology is being re-defined herein to berestricted to including any specific characteristics of the features oraspects of the invention with which that terminology is associated. Thescope of the invention should therefore be construed in accordance withthe appended claims and any equivalents thereof.

1-7. (canceled)
 8. A method for producing a molasses extract comprisingthe steps of: a. diluting the molasses to produce a 10 to 40% w/vaqueous solution; b. optionally filtering the diluted molasses producedin step (a); c. contacting the diluted molasses with a hydrophobicpolymeric adsorbent under conditions sufficient to enable binding ofhydrophobic compounds to the adsorbent and flow through of all othercompounds in the diluted molasses; d. optionally passing the flowthrough from step (c) over the hydrophobic polymeric adsorbent at leastonce; e. optionally rising the hydrophobic polymeric adsorbent; and f.eluting the compounds bound to the hydrophobic polymeric adsorbent toproduce the extract; wherein the hydrophobic compounds are eluted with30 to 70% ethanol; and wherein the molasses extract has a high relativeabundance of hydrophobic compounds including polyphenols and wherein theextract comprises: at least 9000 mg catechin equivalent/100 g ofhydrophobic polyphenols in a mixture of free form polyphenols selectedfrom the group consisting of apigenin, catechin, catechin gallate,epicatechin, kaempherol, luteolin, quercetin, tricin, myricetin anddiosmetin; polyphenol glycosides selected from diosmin,tricin-7-O-neohesperidoside, orientin, vitexin,luteolin-8-C-(rhamnosylglucoside), schaftoside, isoschaftoside andrutin; and phenolic acids, selected from caffeic acid, chlorogenic acid,p-coumaric acid, ferulic acid, gallic acid, syringic acid and vanillicacid; one or more trace elements selected from the group consisting ofcalcium, iron, magnesium, manganese, potassium and sodium; protein andother nitrogen-containing compounds; carbohydrates other thanmonosaccharides and sucrose; and less than 2 g of monosaccharides andsucrose per 100 g of extract. 9-14. (canceled)
 15. The method accordingto claim 8, wherein the method does not include a centrifugation step.16. The method according to claim 8, wherein the filtration step of (b)does not include ultrafiltration.
 17. The method according to claim 8,wherein the filtration step of (b) includes filtering the dilutedmolasses produced in step (a) through a microfiltration membrane. 18.The method according to claim 17, wherein the microfiltration includes amembrane with a pore size of between 0.1 to 0.5 micron.
 19. The methodaccording to claim 18, wherein the membrane has a pore size of 0.1micron.
 20. The method according to any one of claim 8, wherein thehydrophobic compounds are eluted with 70% ethanol.
 21. The methodaccording to any one of claim 8, wherein the molasses extract comprisesat least one of diosmin, chlorogenic acid, and syringic acid.
 22. Themethod according to any one of claim 8, wherein the molasses extractcomprises at least 18000 mg catechin equivalent/100 g of hydrophobicpolyphenols.
 23. The method according to claim 22, wherein the molassesextract comprises at least 21000 mg catechin equivalent/100 g ofhydrophobic polyphenols.
 24. The method according to any one of claim 8,wherein the molasses extract has less than lg of monosaccharides andsucrose per 100 g of extract.
 25. The method according to any one ofclaim 8, wherein the hydrophobic polymeric adsorbent is a polystyreneadsorbent.
 26. The method according to any one of claim 8, wherein thehydrophobic polymeric adsorbent is a food grade adsorbent.
 27. Themethod according to any one of claim 8, wherein the hydrophobicpolymeric adsorbent is Amberlite XAD16 or FPX66.
 28. The methodaccording to any one of claim 8, wherein the method includes thefiltration step (b) and no other pre-treatment steps prior to step (c).29. A method according to any one of claim 8, wherein step (c) comprisesloading the diluted molasses on to the hydrophobic polymeric adsorbentat a flow rate of 2 to 6 L/min.
 30. A method according to claim 29,wherein the flow rate is 4 L/min.
 31. A method according to any one ofclaim 8, wherein in step (c) the diluted molasses is in contact with thehydrophobic polymeric adsorbent for a period of 30-120 minutes.
 32. Amethod according to claim 31, wherein in step (c) the diluted molassesis in contact with the hydrophobic polymeric adsorbent for at least 60minutes, at ambient temperature.
 33. A method according to any one ofclaim 8, wherein in step (d) the flow through from step (c) is passedover the hydrophobic polymeric adsorbent once or twice.